Cardiovascular disease is the most common cause of mortality in adults, and congenital heart defects are the most common form of birth defects in the US. AMP-activated protein kinase (AMPK) is a master regulator of energy balance and homeostasis and plays a central role in the switch from fatty acids to glycolysis during cardiac hypertrophy and heart failure. AMPKa2 is the predominant isoform of the catalytic subunit expressed in the heart and is encoded by the Prkaa2 gene. Although AMPK and its role in metabolism and heart failure have been extensively studied, the transcriptional control of the genes encoding AMPK subunits is completely unknown. The MADS box transcription factor MEF2C is required for cardiac development and functions as a signal responsive transcription factor that interacts with a variety of cofactors to either negatively or positively regulate transcription. The most potent transcriptional coactivator for MEF2C is the SAP domain transcriptional regulator Myocardin. MEF2C specifically interacts with Myocardin-93S, which is highly enriched in the heart, yet the transcriptional targets and mechanisms of synergy facilitated by the Myocardin-MEF2 complex have not been identified. Preliminary studies found that the Prkaa2 gene is a direct transcriptional target of the MEF2C-Myocardin complex via a novel cardiac- specific enhancer. This enhancer contains two bona fide MEF2 binding sites that function together in a multiplicative fashion in response to Myocardin-93S and MEF2C. Although transcriptional synergy is frequently observed in many contexts and is a well-described phenomenon, the role of cis-acting elements, their position, spacing, and sequence in facilitating synergy have been far less well described. Using transgenic mouse, cell culture, biochemical, and mass spectrometry approaches, this work will define determinants of MEF2-Myocardin transcriptional synergy, will identify additional interaction partners for MEF2C and Myocardin through interactome mapping, and will identify in vivo targets of the Myocardin-MEF2 complex. This work will also determine the upstream regulation of Prkaa2 for the first time. This work may provide additional strategies for manipulating AMPK expression during heart failure.